There is increasing evidence that recombinatorial repair is important in human cells, and likely plays a role in preventing cancer. The yeast and human RAD51 genes encode strand-transfer proteins involved in recombination and DNA repair. In. S. cerevisiae, the Rad51 family of related proteins also includes Rad55 and Rad57, which form a heterodimer that interacts with Rad51. In human cells, six proteins in this family (HsRad51, XRCC2, XRCC3, Rad51B/hRec2, Rad51C and Rad51D) likely participate in recombinatorial repair and in maintaining genomic stability. We identified specific interactions between all of these proteins. These interactions suggest that the human proteins are either part of a recombinosome, or form heterodimers and trimers acting in one or more repair pathways. One hypothesis si that the human Rad51- related proteins, like yeast Rad55 and Rad57, function by physically cooperating with Rad51 in DNA repair. This hypothesis will be tested by determining how these proteins interact, and this information should give us important clues to their functions. Our goals are to more rigorously examine these interactions and to determine their biological significance. Co-immunoprecipitation (co-IP) and cytological localization experiments are proposed to extend our yeast two-hybrid results. HsRad51 focus induced by DNA damage will be characterized to see if they contain other Rad51-related proteins. Gel filtration studies will determine if these is one large complex or distinct hetero- multimers . Modified yeast two-hybrid experiment swill determine which pairs of protein interactions can occur simultaneously, and which occur competitively or cooperatively. A major strength of two-hybrid results is that is they rapidly suggest models that will then be tested by co-IP, gel filtration and immunolocalization experiments. The combined data will tell us whether or not these proteins form a recombinosome and if so, will elucidate its architecture. Two-hybrid deletion analysis will be used to map binding domains on XRCC2, XRCC2 and Rad51C, and interaction- defective mutations will be isolated. These interaction-defective mutations will be tested to see if they can complement knockout mutations and/or if they may act as dominant negative alleles. An interaction between Rad51C and IKK-beta has been observed in the two-hybrid system. IKK-beta is a kinase involved in the activation of NF-kappaB, and is part of a system that responds to x-rays and other stimuli. We will attempt to biochemically confirm the observed Rab51C-IKK-beta interaction, and also determine if Rad51C is phosphorylated. If so, we will test if phosphorylation changes in response to x-ray or MMC treatment, and examine its biological significance by analyzing mutant Rad51C that may not be phosphorylated on key residues.